Various publications, including patents, published applications, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference herein, in its entirety. Full citations for publications not cited fully within the specification are set forth at the end of the specification.
The complement system is a key part of the innate and adaptive immune system and plays a major role in homeostasis by clearing altered host cells and invading pathogens (Carroll, 2004; Walport, 2001). Inappropriate activation of the complement system leads to tissue injury causing or aggravating various pathological conditions, such as autoimmune diseases, burn injuries, Alzheimer's disease, stroke and heart attack, among others (reviewed by Sahu and Lambris, 2000). Several complement inhibitors currently under development target various steps in the complement activation pathways. To date, none of these compounds has been approved for clinical use (Bureeva et al., 2005; Holland et al., 2004; Sahu and Lambris, 2000). Many of the substances under investigation possess the disadvantage of being a large molecular weight proteins that are difficult to manufacture and must be administered by infusion. Accordingly, ongoing research continues to emphasize the development of smaller active agents that are easier to deliver, more stable and less costly to manufacture.
U.S. Pat. No. 6,319,897 to Lambris et al. describes the use of a phage-displayed combinatorial random peptide library to identify a 27-residue peptide that binds to C3 and inhibits complement activation. This peptide was truncated to a 13-residue cyclic segment that maintained complete activity, which is referred to in the art as compstatin (SEQ ID NO: 2J. Compstatin inhibits complement response by preventing the proteolytic activation of C3 (Sahu et al., 1996). Activation of C3 by the C3 convertases is a central amplification step in complement activation. All three recognition and initiation pathways, the classical (CP), lectin (LP) and alternative (AP) pathways, converge in the activation of C3. Proteolytic activation of C3 yields C3b, which covalently binds to pathogenic or self surfaces providing a strong signal for clearance of the tagged particles. Because compstatin blocks this critical step of complement activation and because it is a small non-immunogenic peptide, compstatin has the potential to be developed into a therapeutic agent.
Compstatin (SEQ ID NO:2), a 13-residue peptide, circularized by disulfide bond (Cys-2-Cys-12), displays an inhibitory activity of IC50=12 μM. In solution, compstatin forms a β-turn at residues Gln-5-Gly-8 with the disulfide bridge Cys-2-Cys-12, residues Ile-1-Val-4 and Thr-13 forming a hydrophobic cluster (PCT Pub. No. WO99/13899; Morikis et al., 1998; Morikis et al., 2002). Mutational studies showed that the polar 3-turn and the hydrophobic cluster are essential for the inhibitory activity of compstatin (Furlong et al., 2000; Morikis et al., 1998; Morikis et al., 2002; Soulika et al., 2003). Both main-chain and side-chain atoms of compstatin are thought to be involved in interaction with C3 (Sahu et al., 2000). Recently, an analogue of compstatin with 45-fold higher potency was identified, which contained an acetylated N-terminus and amino-acid substitutions V4W and H9A (PCT Pub. No. WO2004/026328; Katragadda et al., 2004; Mallik et al., 2005) (SEQ ID NO: 1). These compounds bind C3 (Kd of 1.3 μM and 0.14 μM for natural compstatin with an acetylated N-terminus and the V4W/H9A analogue respectively (Katragadda et al., 2004)) and its derived products C3(H2O), C3b and C3c (Sahu et al., 1996; Sahu et al., 2000). Soulika et al. (Soulika et al., 2006) showed that the binding site resides in the 40-kDa C-terminal part of the 13-chain that is common to these proteins. Overall, these and other studies have led to a model in which compstatin inhibits complement activation by blocking binding of C3 to the C3 convertases, either through inducing a conformational change in C3 or causing steric hindrance when bound to C3 (Morikis et al., 2002; Soulika et al., 2006).
Additional structural information about C3 has become available recently (Fredslund et al., 2006; Janssen et al., 2006; Janssen et al., 2005; Nishida et al., 2006; Wiesmann et al., 2006). C3 (NCBI Protein Data Bank (PDB) Accession No. 2A73) is a two-chain molecule consisting of a β chain (res. 1-643; Ref. No. 2A73-A; SEQ ID NO:3) and an α-chain (res. 650-1,641) of 75 and 110 kDa respectively that are arranged in thirteen domains, whereas C3c (NCBI PDB Accession No. 2A74) consists of three chains: the β-chain (Ref. No. 2A74-A; SEQ ID NO:4) and two fragments of the α-chain, which form ten domains (Janssen et al., 2005). Activation of C3 occurs by cleavage of the scissile bond Arg-726-Ser-727, generating C3a (9 kDa) and C3b (176 kDa) (Bokisch et al., 1969). The transformation of C3 into C3b induces large conformational changes in the α-chain (Janssen et al., 2006; Wiesmann et al., 2006). In contrast, the β-chain is overall structurally stable. The only exception is the third macroglobulin (MG3) domains, which is part of the MG-ring of the β-chain and shows a reorientation up to 15° when changing from C3 to C3b and C3c (Janssen et al., 2006; Janssen et al., 2005; Wiesmann et al., 2006). The 40 kDa C-terminal fragment, identified by Soulika (Soulika et al., 2006), forms part of MG3 and complete MG4, MG5, MG6β and the linker (LNK) domain (Janssen et al., 2005). Thus, compstatin likely binds to the structurally stable part of C3.
As described above, the solution structure of compstatin, combined with experimental determinations of biological effect, have led to the development of analogs with improved complement inhibiting activity. However, a preferred situation in rational drug design is to have knowledge of the target protein structure along with bound ligands. The more available knowledge, the better the chances of designing and optimizing ligands to modulate therapeutic targets. Accordingly, the elucidation of the structure of compstatin bound to C3 is needed to facilitate rational drug design and to design pharmacophores and identify molecules with even greater activity and desirable biological features. The present invention satisfies this need.